Field of the Invention
An object of the invention pertains to biotechnology, namely a strain producer of plasmid DNA, a relevant pharmaceutical composition which is able to induce growth of blood vessels (vascularization) in the injection site and its application in complex therapy for atherosclerotic lower limb ischemia, as well in treatment of wounds and ulcers of various genesis, and a method for storage of purified plasmid DNA.
Discussion of the Background
Gene therapy with naked plasmid DNA for treatment of diseases or vaccination against pathogens or antigens of tumor cells enables development of finished dosage forms of therapeutic DNA which may be stored, shipped, and used by specialists under unfavorable conditions, and in particular at positive or increased temperatures. Physical and chemical stability of the stored plasmid DNA is significantly defined by a composition of excipients, its concentration and/or the content in the finished dosage form and/or storage conditions (Schleef M., Schmidt T., Animal-free production of ccc-supercoiled plasmids for research and clinical applications, J. Gene Med., 6 Suppl 1:S45-53 (2004)). The main process which affects a pharmaceutically active form of the stored plasmid DNA is degradation of a DNA chain which leads to the formation of relaxed circular plasmid DNA and, subsequently, of a linear two-chain form. It is known that when a frozen plasmid DNA solution is stored at a temperature below minus 80° C., degradation of the supercoiled DNA form is practically unnoticeable (Walther W., Stein U., Voss C., Schmidt T., Schleef M., Schlag P. M., Stability analysis for long-term storage of naked DNA: impact on nonviral in vivo gene transfer, Anal Biochem., 318(2):230-5 (2003)). A such method of storage of plasmid DNA cannot be widely used in clinical practice, as in most cases, healthcare institutions and pharmacies do not have necessary refrigeration equipment. It is rather difficult to ship finished dosage forms and maintain such a low temperature of the product. The selection of appropriate compositions and concentrations of excipients may result in a plasmid DNA solution which is stable for 12 months if stored at 4° C. or over three years if stored at 20° C. (Przybylowski M., Bartido S., Borquez-Ojeda O., Sadelain M., Riviere I., Production of clinical-grade plasmid DNA for human Phase I clinical trials and large animal clinical studies, Vaccine, 25(27):5013-24 (2007)).
The most prevalent method for development of more stable dosage forms is lyophilization. As a rule, lyophilized dosage forms are stable at 4° C. for several years, and in some cases, a product may be stored at room temperature for several months or even two years. Lyophilization also allows to change a concentration of an active substance, wherein vials may be filled with a small amount of a concentrated solution, and the volume may be increased up to the necessary level for product dissolution. Lyophilization of hypotonic solutions and subsequent dissolution of the lyophilizate with a saline solution or a solution with normal osmolality may be used (Anchordoquy T. J., Armstrong T. K., Molina M. C., Low molecular weight dextrans stabilize nonviral vectors during lyophilization at low osmolalities: concentrating suspensions by rehydration to reduced volumes, J Pharm Sci., 94(6):1226-36 (2005)).
Usually, lyophilization of plasmid DNA solutions increases product stability for further storage, but freezing and vacuum sublimation procedures may significantly impair the supercoiled structure of plasmid DNA (Anchordoquy T. J., Armstrong T. K., Molina M. C., Allison S. D., Zhang Y., Patel M. M., et al., Physical stabilization of plasmid DNA-based therapeutics during freezing and drying, In: Costantino H R, Pikal M. J., editors. Lyophilisation of biopharmaceuticals, AAPS press; pp. 605-41 (2004)). In particular, DNA lyophilization from a frozen aqueous solution not containing excipients causes elimination of coordinated water molecules, i.e., a hydrated envelope of a DNA molecule that results in the loss of structural integrity of the DNA molecule (Poxon S. W., Hughes J. A., The effect of lyophilization on plasmid DNA activity, Pharm Dev Technol., 5(1):115-22 (2005)). Consequences of the impaired conformation of a DNA molecule which include degradation of complementary bonds between nitrogen bases and partial degradation of stacking result in undesirable events, i.e., decrease of a biological activity of plasmid DNA up to 25% from the original activity (Anchordoquy T. J., Armstrong T. K., Molina M. C., Low molecular weight dextrans stabilize nonviral vectors during lyophilization at low osmolalities: concentrating suspensions by rehydration to reduced volumes, J Pharm Sci., 94(6):1226-36 (2005)). It is known that a loss of coordinated water molecules eliminated by sublimation may be compensated by the addition of non-volatile hydrophilic substances such as sugars and polyoles to the lyophilized solution (Maitani Y., Aso Y., Yamada A., Yoshioka S., Effect of sugars on storage stability of lyophilized liposome/DNA complexes with high transfection efficiency, Int J Pharm., 356(1-2):69-75 (2008)). A large part of known methods in the field of DNA stabilization by lyophilisation uses freeze-dried lyposomes containing DNA (U.S. Pat. No. 7,323,297), and, therefore, the applicability of know solutions for a solution containing naked plasmid DNA is questionable. In the study of Quaak S., Haanen J., Beijnen J., and Nuijen B., Naked Plasmid DNA Formulation: Effect of Different Disaccharides on Stability after Lyophilisation, AAPS PharmSciTech, Vol. 11, No. 1 (March 2010)), the effect of several polysaccharides on lyophilization of plasmid DNA was examined, and it was established that sucrose which is used with DNA in the mass ratio of 20:1 provided the most stable dosage form for storage. The study did not consider the effect of the pH and a saline solution on stability of plasmid DNA, did not study compositions of isotonic DNA solutions at concentrations below 5 mg/ml, and did not study monosaccharides which are potentially usable for obtaining stable lyophilized macromolecule preparations.